MTT assay

Hello, i am Stanley and this is my second blog posting.

The MTT assay is a quantitative colorimetric method used to determine cell proliferation. It utilizes the yellow tetrazolium salt which is metabolized by mitochondrial succinic dehydrognase activity of proliferating cells to yield a purple formazan product. This purple formazan is then solubilized and the concentration is determined by reading under an optical desity which ranges from 500nm to 600nm. As the MTT assay only detects living cells, it can be use to measure cell cytotoxicity, proliferation or even activation.

Part of my experiment will include the use of MTT assay to determine both the growth curve of both normal lung cell (MRC-5) and large lung carcinoma cells (H460) which are seeded in a 96 well flat bottom plate. The advantages of using MTT will include high accuracy and the exclusion of using radioisotope.

Below are steps for performing the MTT proliferation assay:

Preparation of MTT stock

1) Adding 1ml of sterile PBS into the 5mg vial of MTT

2) Votex the mixture till all of the MTT is dissolved (takes approximately 3 to 5mins to dissolve completely)

(Note: the MTT stock can be stored for four weeks at 4 degrees celsius protected from light)

Labeling of cells


(Before adding the MTT stock)


3) Add 10µL of MTT stock to each well ( The wells will turn to a slight yellow in colour)

4) Incubate at 37 Degrees Celsius and 5% carbon dioxide concentration for 4 hours (1st incubtion)

5) After incubating the plates for 4 hours, remove all but 25 µL medium from each well

6) Add 50µL of DMSO into each plates

7) Incubate the plate for the second time at 37 Degrees Celsius and 5% carbon dioxide concentration for 10mins (2nd incubation)


(After the second incubation)


Absorbance reading

8) Read the absorbance at 540nm using a microplate absorbance reader

The above steps is a quick alternative to the conventional steps which uses SDS sodium dodecyl sulfate - HCL solution instead of DMSO to solubilize or dissolve the purple formazan. However, the conventional steps will require an addition of 4 to 18 hours of incubation time ontop of the first incubation stated in step 4 and has to be read at 570nm instead of 540nm.

Some important points to note are that:

1) DMSO is a powerful solvent and is able to rapidly penetrate the skin and it is important to avoid skin contact with DMSO

2) Both DMSO and the MTT stock are sensitive to light and must be kept away from light to prevent degenerative effects or inaccuracy

Thank You

slide making

hihi, im going to share about slide making of blood specimen for chromosome study.

when the blood culture is ready for harvesting...

1. centrifuge the tube of cell suspension at 1200rpm for 10minutes

2. remove the supernatant and resuspend the cell pellet with fresh fixative
[do not cause too much mechanical disruption to the cells]

3. centrifuge the tube at 1200rpm for 10minutes

4. place the tubes ready for slide making in a rack and prepare a clean recycle tube with pipette for each tube in a rack

5. remove the supernatant into the recycle tube, leaving a suitable amount of fixative to resuspend the cell pellet

6. take a slide from a beaker in the fridge and flick to remove excess water leaving an evenly thin coat of water on the surface of the slide.

7. hold the pipette approximately 1-2 inches above the slides
[tilt the slides at about 45 degree angle]

8. gently drop the cells suspension on the slide

9. dry the sides and edge of the slide and leave it at room temperature to dry

10. check the mitotic index and spread length under the microscope

11. bake the slides at 90 degree celsius for approximately 1 hour 30 minutes


Factors affecting slide making

a) Proportion of fixative
if the metaphases does not spread well, use a higher proportion of acetice acid to methanol. this will soften the cell "sac", thus chromosome spread is better. however, this must be handled carefully as increase amount of acetic acid can cause staining problem later on.

b) Cell density
if the cell density is low, make a denser cell suspension by resuspending the cells with a lesser volume of fixative and vice versa.

c) Temperature of slides
warm slides allow more spreading, thus use it when the spreading is tight. cold slides prevent over spreading of the chromosome. however, cold but dry slides is used to prevent further spreading of the chromosomes. it is normally use when cells are "severely broken".

d) Aging of slides
slides can be sged at 90 degree celsius for 1hour 30minutes or 60-65 degree celsius ovenight.

e) Angle of slides
the angle of the slides is adjusted according to the condition of the metaphase. for example, a steeper angle will create a greater flow direction, thus enhanced the spreading.

by,
yeo sok kian jocelyn
0703359j

Staining

This method of staining is called immunostaining. It makes use of antibodies, primary and secondary.

This is a rather simple but tedious task. The steps are:

1. Fix cells using 100% ethanol. (This is to prevent the cells from washing off, during the wash step)

2. Incubate for ~20 minutes in the freezer.

3. Remove ethanol and wash thrice using PBS.

4. Add 4% blocking solution and incubate for 1hour on rotator at room temperature. (Blocking solution used here is just BSA, bovine serum albumin)

5. Wash thrice using PBS to wash off any unbound BSA.

6. After 1 hour, add primary antibody (Dilution ratio--> Ab: BSA = 1:100) and incubate on rotator for 1hour at room temperature/incubate overnight in freezer.

7. Wash thrice with PBS to wash off any unbound primary Ab.

8. Add secondary Ab (Dilution ratio--> Ab:PBS = 1:100) and incubate for 1 hour on rotator at room temperature.

9. Wash thrice with PBS.

10. Add DAPI and incubate for 5mins on rotator at room temperature.

11. Wash thrice and proceed to view under fluorescent microscope.

Note: DAPI stains for DNA(Nucleus). It will appear blue under microscope.

The antibodies that i use are:

1. Rabbit anti-ZO-1 (Primary Ab)

2. Alexa Fluor 594 goat anti-rabbit IgG (Secondary Ab)

Rabbit anti-ZO-1 will bind to the ZO-1 protein. This ZO-1 protein can be found only when the cells are in contact with each other.

Alexa Fluor 594 goat anti-rabbit IgG is derived from goat and binds specifically to rabbit Ab. Therefore, when the above two Ab are used together, Alexa Fluor 594 goat anti-rabbit IgG will binds to rabbit anti-ZO-1. When view under microscope, the protein will appear red.

Below are some of the photos i taken using the microscope:

Note: Permission were taken from my mentor before the posting of these images, except for the last image. The last image was taken from website: http://www.biotechniques.com/BiotechniquesJournal/2007/August/Murine-nasal-septa-for-respiratory-epithelial-air-liquid-interface-cultures/biotechniques-43042.html?pageNum=4

The yellow box shows the ZO-1 protein that I'm talking about. The reddish thing is the cytoplasm, which is overstained. But for the image below, the distinct red lines are the ZO-1 proteins. The green stain is type IV beta-tubulin stain, according to the website.

Reference:

BioTechniques, 2009. Retrieved on 27 August 2009 from website: http://www.biotechniques.com/BiotechniquesJournal/2007/August/Murine-nasal-septa-for-respiratory-epithelial-air-liquid-interface-cultures/biotechniques-43042.html?pageNum=4

Alvin

Hematology (:

Hey people! This is Siew Ming here to post again (:
I am going to post about my experience when I was attached to the Hematology lab.

Basically, the hematology lab is being divided into two sections: 1) Stat lab where emergency samples were being handled and 2) Routine lab whereby routine processing of blood samples are carried out.

I got the chance to observe and participate in the various tests that are carried out in the lab, such as FBC, ESR, reticulocyte Test, Kleihaur Batek Test and APPT/PT. Besides these, I felt fortunate to be taught to recognize and differentiate the different types of blood cells under the microscope by a very senior med tech! (:

Today, I’m going to share about the techniques of Erythrocyte Sedimentation Rate (ESR). ESR is a screening test used to detect inflammation. However, confirmatory tests have to be carried out to confirm the diagnosis. ESR refers to the rate at which the red cells sediment over time. It is often measured in mm/H. ESR will increase when there is infection, pregnancy, SLE, Anaemia, etc.

For a single ESR test, a minimum of 350ul of venous blood will be collected in an EDTA tube. Before performing the test, it is very crucial to check for visible clots of the blood. This is because clotted samples cannot be processed and the results will be inaccurate and not valid.

Steps:
1. EDTA blood sample is mixed well by inverting the tube several times.
2. 320ul of blood from the EDTA tube is transferred into a Aquisel Tube (filled with Trisodic Citrate 0.106M)
3. The Aquisel tube is then mixed for at least 12 times.
4. A pipette is then introduced into the Aquisel tube through a twisting the pipette in a circular motion, and gradually push the pipette downwards to allow blood to fill the pipette. The blood level should reach the “0” marking on the pipette.
5. The pipette, together with the Aquisel tube is left to stand on a Styrofoam Aquisel Tube holder for 50 mins.
6. Results (no. of mm the cells have fallen) were read at the end of 50min.
7. Results were then recorded into the ESR record book, patient’s request form and LIS verification system.


The expected ranges should be:
Neonates (1-2days): 0-4
Neonates (3 days-1 month), children (2 months – 12 years), and adults (more than12 years): 0-10

I think there is something wrong with blogger these few days. As it doesn’t allow me to post pictures, here’s a link to show how do Aquisel tubes and pipette look like: http://www.swissvacuum.com/products/selecta/Biology_and_histology_equipment/DivisionAnalytique.pdf

Culturing MRC-5 cell lines

Hello, i am Stanley and i will be doing my very first blog posting. I am sorry for the delay as i have only started my experiment this week. I have managed to perform some simple experiment today and i will like to share my experience with you guys. These experiments were done in a bio safety level 2 laboratory and are related to Mammalian cell technology (MCT).



The aim of major project is to evaluate the effect of green tea on large lung cancer cells. I am given two different cell lines which are namely the MRC-5 cell line ( healthy lung cell) and the NCI-H460 ( large cell lung cancer). Both cell lines are previously purchased from ATCC and are both in passage number 2 when they are given to me. The objective for today's experiment is to culture both cells in their respective media therefore allowing healthy cell growth and expansion.



First of all, i will have to prepare the respective media for each cell lines. I will be stating the steps involved in preparing the modified Dulbecco's modified eagle media (DMEM), which will be use to culture the MRC-5 cells.


The steps to prepare 1 litre of DMEM are:

1) Thawing of the Fetal bovine serum(FBS), L glutamin, penicillin streptomycin, sodium pyruvate
in a 37 Degree Celsius water bathe.

2) Dissolve 1 packet of DMEM powder and 37g of sodium bicarbonate into of 1 litre ultra pure
water.

3) Add 10ml of penicillin streptomycin

4) Add 10ml of L- glutamin

5) Add 10ml of sodium pyruvate

6) Add 50ml of FBS

7) Filter sterilize



Although the above steps might seem simple and rather straight forward. I made a mistake of introducing too much bubbles which resulted in overflowing of media into the electric vacuum. Although, i had learnt how to filter sterilize DMEM media in Mammalian cell technology. The modified DMEM media is completely different as it contains 10% FBS and many other constituents. I later learnt from my supervisor that the presence of FBS is the main reason behind the large production of bubbles.

Following the preparation of DMEM media, i am ready to culture my MRC-5 cells. The steps are as followed:

1) Pipette a small amount of DMEM media into a centrifuge tube

2) Pipette 2 tubes of MRC-5 cell line into the centrifuge tube

3) Centrifuge it at 1000 rpm for 5mins

4) Pipette out the supernatant

5) Add 20ml of DMEM media to resuspend the cells

6) Pipette the resupended cells into a T75 flask
There are a few key points to note while culturing cell
7) Incubate the culture under 5% Co2 level at 37 Degrees Celsius



While culturing fragile cells for example the MRC-5. We will have to ensure that the media is properly warmed to around 37 Degrees Celsius which is near to our body temperature before it can use to culture the cells. Similarly, no bubbles should be present in the culture as this might lead to cell death.


Strict aseptic techniques have to be observed throughout the whole experiment as any contamination will results in the discarding of media or materials. It is rather scary as my supervisor had previously ordered the materials in week 2 but the it only arrive on week 6 of my major project.

Here are how both cells look like under an inverted microscope.

Mrc-5 cells--------------------->

H460 cells ----------------->

Thank you

Islet isolation (:

Hi! I'm back for episode 2!

Isolation of pancreatic islets from mice

Retrieved from BBC (2008). Scientists make weight loss claim. Retrieved on 01st July 2009, from: http://news.bbc.co.uk/2/hi/health/7372495.stm

For this experiment you will need these few things:
White mice, Anaesthesia, Surgical Scissors, surgical wire, Forceps, Syringe, Needle, Tube, Collagenase solution.

When I first stepped into the animal room, a strong smell engulfed me. At first I thought it smelt nice (maybe because it was before lunch), but as I babysit the mice longer, they started to stink. The mice were stored in a styrofoam with holes as they were transported from a facility (which I shall not name).

A total of 4 mice were used in this experiment. The mice were making a lot of noise and they kept moving around! They almost had the strength to push open the styrofoam cover and even tried to escape.

My senior picked up the mouse by its tail, and tried to hold up its head by its ears in between her thumb and index fingers and held the tail using her last finger. Basically, the mouse was immobilized by a hand. The other hand was used to inject the anaesthetic into the mouse’s abdomen. This has to be done carefully. If injected wrongly, this may cause the mice to die earlier, which will be no good for the experiment.

As you guys know, mouse is very small. Therefore my mentor used a binocular microscope for easier visualization. To ensure that the mouse is really asleep, she will pinch its legs to check for reaction. No reaction would indicate the mouse is asleep. Next, she used a surgical scissors to cut open the abdomen, and exposed its contents.

Do you know that a mouse’s liver is really big? At first, I thought the liver was the lung! Below the liver is the pancreas. First, my mentor skillfully tied up the bile duct with the surgical wire. Tying up the bile duct before injecting collagenase into the mice ensures that collagenase will only be injected in the pancreas. If not, collagenase solution will flow into the stomach and intestine, therefore the pancreas will not swell up. Next, she carefully inserted a small tube which contains collagenase into the bile duct. Collagenase is used to digest the external cells. Once collagenase is pumped into the pancreas, the pancreas starts to swell up. I once thought that the pancreas is green in colour, but actually it is quite yellowish-transparent. Once the entire amount is injected in, the tube was carefully removed and pancreas was removed and placed in a tube containing collagenase. The tube was then placed in 37*C waterbath. After the removal of pancreas, the mouse would die a few minutes later.

However, my observation did not stop here. As my senior needed to collect blood for her experiment, I watched as she inserted the needle into the mouse’s really small heart and took the small little amount of blood. It was really weird, as during the procedure, the mouse would have reflex action, almost like the mouse is having hiccups.

Lastly, after we have gotten what we needed, the mice were covered in aluminium foil for disposal.

after putting pancreas in collagenase, you'll incubate at 37oC for 17-18 minutes. after that, you'll shake the cells, to ensure homogenicity and check that there is no remnants. next, we will pour the solution into a petri dish. using a binocular microscope, islet cells are handpicked.the cells will be transferred into a petri dish containing RPMI (Roswell Park Memorial Institute) media which is suitable for islet growth. the cells. the handpicking of islet cells are repeated onto subsequent petri dishes to remove all the epithelial cells.
Although I wouldn't have a chance to ever cut the mouse open, but this has been a great experience, though this isn't and will not be my first and my last.

Lim Jia Hui (JOEY)
0703605F

Microbiology - questions asked

hihi.. sorry for the late reply. i didnt know the response was so overwhelming. hahaha..

please read on.... =)

to Jess

Question: does Salmonella appear black on all agar

No. it appear black only on XLD (Xylose lysine deoxycholate) plate.

Question: how does E.coli and Acinetobacter baumanii appears on the agar that differs from Salmonella

Both Salmonella and Acinebacter baumanii is non-lactose fermenter. Whereas E.coli is a lactose fermenter. Therefore Macconkey plate can be used to differentiate the lactose fermenter from the non-lactose fermenter. Inorder to differentiate the three different types of bacteria, perform biochemical test.

to Siewming

Question: what is Tip specimen

when patient with dextrose drip, central venous catheter, or any form of needle attaching to body, shows signs and symptoms of infection (eg. fever), blood FEME is usually done. when the blood FEME shows that there is bacteria growth or high amount of WBC present,

it means positive for infection. Hence it might be due to tip contamination, which is contamination of the needle or even the tubing. Thus, part of the tube is cut and send for comfirmation.

Question:is there a need to culture different type of stool on different type of agar plate.

Yes. different form of stool formed is due to different bacteria. Hence, with a standard protocol in the lab, different type of agar plate is used so to select and maximise the growth of the target bacteria.

to Jordan

Question: is it a must to streak on TSA plate then Macconkey plate for urine specimen.

Yes, Macconkey agar is a selective agar which may inhibit the growth of certain organism. thus if we streak on Macconkey agar first, we might transfer some of the nutrient in the Macconkey to the TSA plate which may inhibit the growth of certain organism. this may lead to inaccurate result as there might be more than 1 organism present.

to Jeremy

Question: what does XLD stands for and its uses in stool culture

XLD stands for Xylose Lysine Deoxycholate agar. Basically it is use to isolate Salmonella and Shigella.

Question: why does Hemophilus Influenza (HI) unable to grow on blood agar and is there any way to establish growth of HI on blood agar

HI does grow on blood agar only with the presence of Staphylococcus streak. this is bacause, Staphlococcus streak contains the X and V factor with enhance the growth of HI.

Question: what is the significance of detecting MRSA in people.

The only antibiotic that can treat MRSA is vancomycin. Given that vancomycin will damage the kidney function of neonates, it is very crucial.

to Lok Pui

Question: why do we need to do cell count for urine specimen

FEME is done to support the report. For instance if there is bacteria growth on the agar plate but there is no WBC observe under the microscope, it could be due to contamination of the agar plate. Hence urine FEME serves as a countercheck for the results.

Question: how to culture solidfy stool

Solid stool doe not undergo any dilution in broth. A stick is use to scrap abit of the stool, then it is smear on the agar plate.

Question: what kind of specimen is used for ear culture

For ear culture, ear swab is obtain.

(extra info: ear culture is done more commonly in newborn to screen for Group B Streptococcus that may be pass on from the mother.)

to Liyana

Question; how germ test is performed to confirm for Candida Albicans

In germ test, rich plasma is supplied. Only Candida Albicans is able to grow germ tube within 24 hours.

this is a picture of germ tubes of Candida Albicans

(adapted from http://en.wikipedia.org/wiki/Germ_tube)

to Janice

Question; do we use different plate for different form of stool

Yes, we do use different plate for different form of stool. (for more information, can refer to the post that i wrote)

Question: do we put ear specimen on cook meat

Nope. we only use CDC and Macconkey without salt agar plate.

Question; for Salmonella found on plate, how do we identify

we do a serotyping to confirm which group of Salmonella it belongs to. In this test, Salmonella Antisera is used. There is a total of 9 different group of Salmonella. In order to perform the serotyping, ensure that bacteria looks homogenous and no autolysis occurs. Next, one colony is treated with PO and PH, a bacteria antigen. PH is a test against flagella . Both PO and PH must be positive to ensure that the bacteria is a Salmonella species. Commercial reagent is then used to test for the group.

for those questions on the chat box, so sorry, i didnt realise there were questions over there. hence its not included in this post. will reply soon !! =)

bye bye..

JOCELYN =D